Targeting the metabolism of tumor cells is now well recognized as a powerful strategy to develop new therapeutics which could improve treatment options especially of tumor types that are resistant to standard chemo-therapy. Most of previous studies have focused on the precept that the mitochondria are dysfunctional in cancer cells and that tumors depend upon glycolysis for growth (the Warburg effect). This proposal provides a paradigm-concept shift from this view, with our discovery that the mitochondrial citrate transporter SLC25A1 (CIC) is up-regulated in many cancers, is necessary for continuous tumor outgrowth, yet supports proliferation by actually promoting mitochondrial function and by blunting glycolysis. We have also shown that CIC is necessary for the survival response that cancer cells mount in order to adapt to restriction of glucose and to mitochondrial damage. These two forms of stress inevitably ensue in the limiting microenvironment due to the irregularity of the vasculature and pose an important obstacle to the expansion of tumor cells. Thus, our data place CIC activity at the core of the mechanisms by which cancer cells acquire a proliferation advantage in these conditions. Our results specifically show that CIC promotes metabolic adaptation by enacting the switch from glycolysis to gluconeogenesis and by enhancing mitochondrial amount and activity. We have also identified two chemical inhibitors of CIC that display anti-tumor activity as single agents and are non toxic in adult mice. Based on these premises this proposal has three objectives. In Aim 1 we will test the idea, supported by our preliminary data, that CIC maintains the homeostatic control of the tumor mitochondria by inhibiting the rates of mitochondrial degradation and by interfering with the mitochondrial division machinery. We show that through these activities CIC promotes an increase of mitochondrial amount during stress conditions that, in the absence of CIC, would instead lead to mitochondrial depletion, thus depriving tumor cells of their power engine. In Aim 2 we will use NMR spectroscopy and metabolic profiling to confirm our preliminary findings that CIC promotes metabolic plasticity by influencing mitochondrial and cytoplasmic pathways of energy production, thus enacting adaptation to stress. In Aim 3 we will exploit pre-clinical mouse models to study the chemo-therapeutic potential of CIC inhibitor compounds. This will be achieved by employing canonical cancer cell lines as well as patient-derived tumor biopsies expanded as conditionally reprogrammed cells (CRC). The emphasis of these in vivo studies will be on lung cancer based on the important observations that high CIC expression levels predict the poorest prognostic outcome in patients affected by this disease and that CIC promotes the rapid outgrowth of lung cancer cells in vivo. The ultimate goal of these studies is to obtain proof of principle that CIC inhibitors will allow the more effective treatment of well-defined and clinically relevant lung cancer sub-types that are otherwise difficult to target with currently available chemo-therapy.